Proper nervous system functioning critically depends on the proper development of an intricate network of neuronal connectivity. It is well established that defects in neural network assembly or interruption of synaptic connections as a result of disease or injury leads to severe neurological deficits. The Semaphorins are one important class of molecules known to regulate axonal growth, guidance, and plasticity. We have identified Semaphorin 5A (Sema5A) as a bi-functional axon guidance molecule that regulates neuronal growth in a proteoglycan-dependent manner in vitro. Specifically, in the presence of chondroitin sulfate proteoglycans (CSPGs), Sema5A inhibits neurite outgrowth, whereas in the presence of heparan sulfate proteoglycans (HSPGs), SemaSA promotes neurite outgrowth. The studies outlined in this proposal directly build on our in vitro findings and are aimed at the functional characterization of Sema5A in specific neuronal populations during nervous system development in vivo. In specific Aim 1, I propose to conditionally ablate Sema5A in the embryonic nervous system in order to study its role during the development of a major limbic fiber tract called the fasciculus retroflexus. I propose to cross our Sema5A conditional mice with specific cre- driver lines to selectively ablate Sema5A in the embryonic nervous system. In specific Aim 2,1 will explore mechanistic questions to gain insights into how Sema5A regulates neuronal growth in a proteoglycan dependent manner. The methods I will use to achieve my goals include the use of state-of-the-art mouse genetics, immunohistochemical and biochemical techniques. To ask mechanistic questions related to Sema5A function, I will employ in vitro membrane stripe assays with primary neurons from wild-type and Sema5A mutant animals. As a whole, the studies proposed are anticipated to provide important insights into how Sema5A functions in the development of specific central nervous system fiber tracts, and importantly, reveal which proteoglycans switch Sema5A-mediated growth to Sema5A-mediated inhibition. If successful, our studies will provide the first mechanistic clues of how CSPGs influence neuronal growth. Lay language summary: Chondroitin sulfate proteoglycans (CSPGs) are large extracellular molecules that are increasingly being recognized as important regulators of nerve cell growth during development. In addition, CSPGs have been implicated in limiting nervous system regeneration following injury. The work ' proposed here is mission-oriented-it is aimed at understanding how CSPGs instruct nerve cells not to grow. We expect to uncover new biological principles that may be relevant for the treatment of developmental disorders and/or promote nervous system repair following injury or disease. [unreadable] [unreadable]